IN VIVO CHEMILUMINESCENT ACTIVATION OF PHOTOSENSITIZERS

说明：金丝桃素是一种自然产生的光敏剂 当被光激活时，显示出强大的抗病毒和抗肿瘤活性。 金丝桃素的抗病毒活性对许多 包膜病毒，包括艾滋病毒-1，艾滋病的病原体。然而， 金丝桃素用于抗病毒或癌症治疗的体内应用受到阻碍 通过它的光依赖性。这些研究的长期目标是产生 化学发光作为体内激活病毒的光源 以及金丝桃素和其他光敏剂的杀瘤活性。这个 这项建议的目的是确定 金丝桃素在细胞内的化学发光激活。化学发光体 萤火虫荧光素酶与荧光素的作用可激活抗病毒作用 金丝桃素在无细胞体系中的活性；然而， 研究发现，能量转移依赖于局部浓度的 能量供给者和接受者。在第一个具体目标中， 能量供体和受体，即荧光素和金丝桃素，将增加 通过化学系绳的方式。第一种类型的束缚分子将是 笼状的荧光素，其中的羧酸基存在于荧光素中 是一种活化酯。第二种类型的束缚分子将 是那些在酯酶存在的情况下保持其完整性的。这 设计将利用非常有效的分子内能量 金丝桃素与金丝桃素化学发光中间体之间的转移 荧光素与荧光素酶的反应。所有被束缚的化合物都将被 初步测试了光化学性质，有希望的分子将 在生物学上有进一步的特征。第二个具体目标，稳定 慢病毒控制下表达荧光素酶基因的细胞系 启动子将被分离，感染病毒，并进行拴系治疗 分子。将使用共聚焦显微镜和荧光显微镜来定位 病毒感染细胞内的金丝桃素纽带和荧光素酶。反作用 荧光素酶和束缚荧光素之间的关系将作为以下函数进行监测 金丝桃素活化的荧光光谱分析。最后，细胞将 被检测是否产生传染性病毒。这些研究的结果 将确定化学发光激活金丝桃素或其他 光敏剂可以在体内应用。拟议的研究代表了一个 多学科方法开发新型抗病毒药物和 抗肿瘤疗法。

DESCRIPTION: Hypericin is a naturally occurring photosensitizer that exhibits potent anti-viral and anti-tumor activity when activated by light. The antiviral activity of hypericin is effective against a number of enveloped viruses, including HIV-1, the causative agent of AIDS. However, in vivo application of hypericin for antiviral or cancer therapy is hampered by its photodependency. The long range goal of these studies is to generate chemiluminescence as an in vivo light source for activation of the virucidal and tumoricidal activity of hypericin and other photosensitizers. The objectiv of this proposal is to determine the feasibility of chemiluminescent activatio of hypericin in cells. The chemiluminescent reaction of firefly luciferase and luciferin can activate the antiviral activity of hypericin in a cell free system; however, the efficiency of energy transfer was found to depend on the localized concentrations of the energy donor and acceptor. In the first specific aim, the proximity of the energy donor and acceptor, i.e. luciferin and hypericin, will be increased by means of a chemical tether. The first type of tethered molecular will be a "caged" luciferin, wherein the carboxylic acid group present in luciferin is capped as an activated ester. The second type of tethered molecules will be those which maintain their integrity in the presenc of esterases. This design will take advantage of a very efficient intramolecular energy transfer between hypericin and the chemiluminescent intermediate of the reaction of luciferin with luciferase. All tethered compounds will be tested initially for photochemical properties, and promising molecules will be further characterized biologically. In the second specific aim, stable cell lines expressing the luciferase gene under the control of a lentivirus promoter will be isolated, infected with virus, and treated with tethered molecules. Confocal and fluorescence microscopy will be used to localize hypericin tethers and luciferase within virus-infected cells. Reactions between luciferase and tethered luciferin will be monitored as a function of hypericin activation using fluorescence spectroscopy. Finally, cells will be assayed for production of infectious virus. The results of these studies will determine if chemiluminescent activation of hypericin or other photosensitizers can be applied in vivo. The proposed studies represent a multidisciplinary approach to the development of novel antiviral and anti-tumo therapies.